PROJECT SUMMARY The basis for the success of radiation therapy is long believed to be direct killing and elimination of cancer cells. Therefore, most previous research efforts have focused on enhancing the direct killing effects of radiotherapy. However, recently, it was realized that the host immune system plays key roles in determining the treatment outcome of cytotoxic cancer therapy. In many instances, it has been shown that radiation therapy can activate the immune system to attack cancer cells. However, the molecular mechanisms involved in radiation-induced immune cell activation are poorly understood. In the current project, we intend to test the hypothesis that blockade of certain apoptotic caspases will enhance the efficacy of radiotherapy and immune checkpoint inhibitors by increasing immunogenic cell death. Our hypothesis is based on both published data on the immune-stimulatory effect of radiotherapy and our preliminary data demonstrating strong immunogenic effects of blocking apoptotic caspases. To test our hypothesis, we will attempt to dissect the roles of individual caspases by use of murine tumor cells with CRISPR-Cas9 mediated genetic knockout and evaluating their response to radiotherapy in vitro and in mice (Aim 1). We will then try to identify downstream factors of caspases that are important in radiation induced immunogenic cell death (Aim 2). Finally, we will evaluate if inhibition of caspases is a feasible strategy to enhance radiotherapy in combination with immune- checkpoint inhibitor therapy (Aim 3). Upon completion of our proposed project, we hope to establish the biological roles of apoptotic caspases in immunogenic cell death during radiotherapy and lay the foundation for evaluating caspase inhibitors as potential agents to enhance radiotherapy and immuno-checkpoint inhibitor therapy in human patients.